In the currently prevailing methods, generally two proteins are cross-linked, using a bi-functional cross-linker, at random sites to a protein residue, e.g., lysine, which is distributed at several places on the protein surface. This method of cross-linking often blocks the functional sites on the protein and thus reduces the bioefficacy of the protein. In contrast, the advantage of the method of linking two glycoproteins or glycoconjugates via glycan residues utilizing glycosyltransferases, described in the project Z01 BC 010742, has specific advantage since the linkage occurs between the two partners at a defined site, where the glycan moiety is attached to the glycoconjugates. For example, a cargo can be attached to a monoclonal antibody at the asparagine-linked glycan chain, which resides in the Fc-fragment, away from the antigen binding site, and delivered to the site of action without altering the bioefficacy of the monoclonal antibody. In this project we are coupling the apoptotic molecules HAMLET, Galectin-1 and Galctin-3, to the therapeutic monoclonal antibodies using the method described in the project Z01 BC 010742. HAMLET (Human Alpha-lactalbumin (HLA) Made to Kill Tumor cell) prepared from recombinant in vitro folded HLA, with or without a C-terminal fusion polypeptide show comparable tumoricidal activities: The biological killer molecule HAMLET, a complex of apo alpha-lactalbumin and oleic acid, has been shown by Dr. Catharina Svanborg to trigger tumor cell death while healthy cells are spared. HAMLET treated tumor cells undergo apoptosis. Since we have been the first to have cloned, expressed and in vitro folded the protein, determined the structure of alpha-lactalbumin in complex with galactosyltransferase and extensively studied the molecule over two decades, we have the expertise to produce the HAMLET and conjugate it to a monoclonal antibody or a single chain antibody at a specific site for its targeted delivery. In the FY 09-11 we have expressed the recombinant human alpha-Lactalbumin without (rhLA) or with a C-terminal fusion polypeptide (rhLA-ext) in E. coli and folded the proteins in vitro and converted each to molten globule protein by removal of Ca2+. The molten globule proteins are complexed with oleic acid to form HAMLET. These HAMLET preparations, and not the molten globule proteins or oleic acids alone, show tumoricidal activity against several human cancer cell lines including SKBR3, MDA-MB68, MCF-7 and A549. Oleic acid complexes derived from rhLA and rhLA-ext had comparable tumoricidal activities as measured by Trypan blue assay and Annexin-V method. Following glycosylation of the fusion polypeptide of rhLA-ext at a unique site with 2-keto-galactose using ppGalNAc-T2, as described in the project Z01 BC 010742. The protein was conjugated with Alexa Fluor 488 via the chemical handle, C2-keto group, of galactose. Using confocal live microscopy, the labeled HAMLET, the complex of labeled molten globule rhLA-ext with oleic acid, and not the labeled molten globule rhLA-ext alone shows staining in the cell membrane, cytoplasm and latter accumulation in the cell nuclei. Thus we have been successful in adding a chemical handle to HAMLET without affecting its biological activity, allowing us to trace the complex at the cell surface or inside the cell. The addition of a site-specific handle to a tumoricidal complex could be further exploited for isolation of partner/target molecules, providing insight into the tumor specificity of the complex. Alpha-domain of rhLA (aD-rhLA) complexed with OA has tumoricidal activity comparable to HAMLET: Alpha-lactalbumin has 4 helices that are contained in the alpha-domain and beta-sheets that form a beta-domain. The alpha-domain of hLA (aD-hLA) with a polypeptide tag at the C-terminal end can be specifically glycosylated with ppGalNAc-T2, transferring a modified galactose with a chemical handle (C2-keto-galactose) that can be further labeled with aminooxy-Alexa Fluor 488. We have now shown that the tumoricidal complex derived from the site-specific-labeled aD-hLA-ext and oleic acid also kills many tumor cells and the labeled protein is internalized in tumor cells. Only aD-hLA-ext protein complexed with oleic acid was internalized after 3 h of incubation. These results indicate that we have been successful in expressing, labeling, and imaging, by confocal microscopy, a smaller form of alpha-lactalbumin complexed with oleic acid, a HAMLET-like molecule that still exhibits tumoricidal activity. The addition of a site-specific handle to a tumoricidal complex could be further exploited for isolation of partner/target molecules, providing insight into the tumor specificity of the complex, the work that is in progress.